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J S Philo

Publications and source records attributed to J S Philo.

At least 37 records · Page 2Linked to original sources
PubMed1996

Human hepatic lipase subunit structure determination.

Chinese hamster ovary cells were stably transfected with a human hepatic lipase (HL) cDNA. The recombinant enzyme was purified from culture medium in milligram quantities and shown to have a molecular weight, specific activity, and heparin affinity equivalent to HL present in human post-heparin plasma. The techniques of intensity light scattering, sedimentation equilibrium, and radiation inactivation were employed to assess the subunit structure of HL. For intensity light scattering, purified enzyme was subjected to size exclusion chromatography coupled to three detectors in series: an ultraviolet absorbance monitor, a differential refractometer, and a light scattering photometer. The polypeptide molecular weight (without carbohydrate contributions) was calculated using the measurements from the three detectors combined with the extinction coefficient of human HL. A single protein peak containing HL activity was identified and calculated to have a molecular mass of 107,000 in excellent agreement with the expected value for a dimer of HL (106.8 kDa). In addition, sedimentation equilibrium studies revealed that HL had a molecular mass (with carbohydrate contributions) of 121 kDa. Finally, to determine the smallest structural unit required for lipolytic activity, HL was subjected to radiation inactivation. Purified HL was exposed to various doses of high energy electrons at -135 degrees C; lipase activity decreased as a single exponential function of the radiation dose to less than 0.01% remaining activity. The target size of functional HL was calculated to be 109 kDa, whereas the size of the structural unit was determined to be 63 kDa. These data indicate that two HL monomer subunits are required for lipolytic activity, consistent with an HL homodimer. A model for active dimeric hepatic lipase is presented with implications for physiological function.

Animals↗
PubMed1996

Size-exclusion chromatography with on-line light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions.

Techniques of using size-exclusion chromatography (SEC) with on-line light-scattering, uv absorbance, and refractive index detectors to characterize the polypeptide molecular weights of simple proteins or glycoproteins or to determine the stoichiometry of protein complexes are described. Two unique advantages of this approach over conventional SEC are that the molecular weight measurement is independent of elution position and can exclude the contributions from carbohydrates. When a protein or complex contains no carbohydrates, a two-detector method, i.e., light scattering combined with refractive index, can be used to calculate the molecular weight. When a protein contains carbohydrates, a three-detector method is used to calculate the molecular weight of polypeptide alone. Finally, a self-consistent three-detector method is used to determine the stoichiometry of a protein complex containing carbohydrates. Example applications for all these methodologies are described.

Carbohydrates↗
PubMed1996

Induction of alpha-helix in the beta-sheet protein tumor necrosis factor-alpha: thermal- and trifluoroethanol-induced denaturation at neutral pH.

The unfolding and refolding of alpha-helical proteins has been extensively studied, demonstrating formation of intermediate structures which retain the native-like alpha-helix but lack the tertiary structure. Studies on the folding of proteins consisting primarily of beta-sheet are interesting since, unlike the alpha-helix, the beta-sheet requires the formation of peptide hydrogen bonds between two or more polypeptide segments which may be far apart in the linear sequence. Here we have studied the unfolding of the beta-sheet-containing protein tumor necrosis factor-alpha (TNF-alpha). This protein exists as a symmetric trimer in solution. Murine TNF-alpha begins to melt at 60 degrees C and unfolds to a soluble structure with a transition midpoint of 66 degrees C. This reaction is irreversible. This unfolded form contains a considerable amount of (approximately 30%) alpha-helix, as determined by circular dichroism. Human TNF-alpha begins to melt at 60 degrees C and precipitates concurrently with unfolding, such that there is no soluble protein present by 70 degrees C. The secondary and tertiary structures of murine TNF-alpha unfold simultaneously, suggesting that unfolding from the native to the unfolded state occurs cooperatively. The thermal-induced denaturation is very insensitive to protein concentration, indicating that trimer to monomer conversion, if it occurs, is not rate-limiting. Trifluoroethanol induces alpha-helix in both human and murine TNF-alpha, further demonstrating the propensity of TNF-alpha to form alpha-helix. The different behavior of human versus murine TNF-alpha upon thermal unfolding is due to differences in the solubility of the unfolded protein, the murine form being more soluble. These results indicate that TNF-alpha can form alpha-helix when the long range interactions conferred by the native structure are removed during unfolding.

Animals↗
PubMed1996

Induction of alpha-helix in the beta-sheet protein tumor necrosis factor-alpha: acid-induced denaturation.

Acid-induced unfolding of proteins often results in an intermediate structure, called the molten globule structure or "A" state, which retains at least partial secondary structure but lacks a rigid tertiary structure. Acid-induced unfolding has been studied extensively for alpha-helical proteins, while few studies have been done on proteins containing only beta-strands. Tumor necrosis factor-alpha (TNF-alpha) is a trimer in which the individual subunits consist of antiparallel beta-sheet, organized into a jellyroll beta-sandwich. We have found previously [Narhi et al. (1996) Biochemistry 35, 11447-11453] that thermal denaturation of TNF-alpha results in an aggregate which contains a substantial amount of alpha-helix and that the addition of trifluoroethanol induces alpha-helix in both murine and human TNF-alpha. Here we show that acid also can induce alpha-helix in these proteins. At acidic pH (below 4), both human and murine TNF-alpha convert to a monomeric form, as determined by sedimentation and diffusion constants obtained from sedimentation velocity experiments. The sedimentation coefficient indicated that this monomer was only slightly expanded relative to the native state. Near-UV circular dichroic (CD) analysis showed a loss of tertiary structure. These structural features coincide with the notion that the acid-induced structure of TNF-alpha is a molten globule. What is unique in this protein is that TNF-alpha acquires alpha-helical structure, which is not present in the native structure as determined by both CD and Fourier transform infrared spectroscopy. Even more surprising is that TNF-alpha at pH 3.3 undergoes a very gradual noncooperative change in secondary structure upon heating, which results in an increase in alpha-helical content. At pH 2.2 in the absence of salt, TNF-alpha shows considerable alpha-helix, although heating does not change the spectrum. At pH 2.2, physiological salt decreases the amount of alpha-helix at ambient temperature, and upon heating, we see the noncooperative increase in alpha-helix as observed at pH 3.3 with low salt. The addition of salt at low pH induces reassociation but to a range of oligomers rather than a unique trimer structure. This acid-induced formation of an alpha-helical monomer of TNF-alpha may be related to its known interaction with lipid bilayers.

Animals↗
PubMed1996

Characterization of keratinocyte growth factor binding to heparin and dextran sulfate.

Binding of keratinocyte growth factor (KGF) with heparin (molecular weight of 5000) and dextran sulfate (molecular weight of 8000) was studied using an online monitoring of size-exclusion chromatography with light scattering, refractive index, and uv absorbance detectors. This technique allows the determination of the molecular weight of KGF eluting as complexes with the above polymers. When mixtures of KGF with heparin were injected into the column, two peaks of heparin/KGF complexes were observed. The first peak corresponded to, on average, 3.4 KGF per complex and the second peak to an average of about 2 KGF per complex. These results suggest that the heparin/ KGF complex is heterogeneous, consisting of 1,2,3, and 4 KGF molecules per complex. To calculate the number of heparin molecules in these complexes, the rate of disappearance of free KGF was determined as heparin was added. The average number of KGF bound to 1 mol of heparin was calculated to be about 2 mol, suggesting that only one heparin molecule is present in these complexes. The heparin binding of two KGF mutants, i.e., (C1, 15S)KGF (with substitutions of serine for cysteines 1 and 15) and d28KGF (lacking 28 N-terminal amino acid residues), was essentially identical to that of the native sequence KGF. A similar experiment was carried out for KGF binding to dextran sulfate. The molecular weight of the complex corresponded to 2 to 2.6 KGF molecules per complex. The rate of disappearance of free KGF as the dextran sulfate added showed 2-3 mol of KGF bound to 1 mol of dextran sulfate, consistent with the idea that the complex contains only 1 dextran sulfate molecule.

Binding Sites↗
PubMed1996

Human stem cell factor dimer forms a complex with two molecules of the extracellular domain of its receptor, Kit.

Stem cell factor (SCF) is a cytokine that is active toward hematopoietic progenitor cells and other cell types, including germ cells, melanocytes, and mast cells, which express its receptor, the tyrosine kinase, Kit. SCF exists as noncovalently associated dimer at concentrations where it has been possible to study its quaternary structure; it stimulates dimerization and autophosphorylation of Kit at the cell surface. We have used recombinant versions of human SCF and human Kit extracellular domain (sKit) to study SCF-Kit interactions. By size exclusion chromatography, plus various physical chemical methods including light scattering, sedimentation equilibrium, and titration calorimetry, we demonstrate the formation of complexes containing a dimer of SCF (unglycosylated SCF1-165) plus two molecules of sKit. The concentrations of SCF and sKit in these studies were in the range of 0.35-16.2 microM. The data are analyzed and discussed in the context of several possible models for complex formation. In particular, the sedimentation data are not consistent with a model involving cooperative binding. The Kd estimate for SCF-sKit interaction, obtained by sedimentation equilibrium, is about 17 nm at 25 degrees C. With glycosylated SCF1-165, the Kd is considerably higher.

Animals↗
PubMed1996

Dimerization of the extracellular domain of the erythropoietin (EPO) receptor by EPO: one high-affinity and one low-affinity interaction.

Although there is considerable evidence that signaling by the erythropoietin (EPO) receptor is initiated when it is dimerized by binding EPO, it has been previously reported that the soluble extracellular domains of the EPO receptor (sEPOR) are not dimerized in the presence of EPO and are able to form only 1:1 complexes with EPO. We have now shown unambiguously by light scattering, sedimentation equilibrium, and titration calorimetry that two molecules of sEPOR can bind to a single EPO monomer but that the binding of the second sEPOR is approximately 1000-fold weaker than that of the first. Because this second binding interaction is quite weak (Kd of approximately 1 microM), the 2:1 sEPOR.EPO complexes are easily dissociated during chromatography (forming the 1:1 complexes reported previously) and cannot be isolated in pure form. Global analysis of the sedimentation equilibrium data has enabled us to determine the binding constants and is consistent with a model in which EPO has two independent binding sites for sEPOR but cannot exclude anticooperative or sequential binding models. The influence of glycosylation of EPO and/or sEPOR on the binding affinities has also been investigated. Titration calorimetry is consistent with the sedimentation data and shows that the weaker binding site has a more negative delta H. The relation of these results to the binding of EPO to membrane-bound receptors and to the phenomenon of apparent high-affinity and low-affinity classes of receptors is discussed.

Animals↗
PubMed1996

Quaternary structure dynamics and carbon monoxide binding kinetics of hemoglobin valency hybrids.

The kinetics of CO binding and changes in quaternary structure for symmetric valency hybrids of human hemoglobin have been extensively studied by laser photolysis techniques. Both alpha+beta and alpha beta+ hybrids were studied with five different ferric ligands, over a broad range of CO concentrations and photolysis levels. After full CO photolysis, the hybrid tetramers switch extensively and rapidly (< 200 microseconds) to the T quaternary structure. Both R --> T and T --> R transition rates for valency hybrid tetramers with 0 and 1 bound CO have been obtained, as well as the CO association rates for alpha and beta subunits in the R and T states. The results reveal submillisecond R reversible T interconversion, and, for the first time, the changes in quaternary rates and equilibria due to binding a single CO per tetramer have been resolved. The data also show significant alpha-beta differences in quaternary dynamics and equilibria. The allosteric constants do not vary with the spin states of the ferric subunits as predicted by the Perutz stereochemical model. For the alpha beta+CN hybrid the kinetics are heterogeneous and imply partial conversion to a T-like state with very low (seconds) R reversible T interconversion.

Allosteric Regulation↗
PubMed1996

Complete sequence, subunit structure, and complexes with pancreatic alpha-amylase of an alpha-amylase inhibitor from Phaseolus vulgaris white kidney beans.

The complete amino acid sequence of a white kidney bean (Phaseolus vulgaris) alpha-amylase inhibitor (PHA-I), which is composed of two kinds of glycopolypeptide subunits, alpha and beta, was established by conventional methods. The polypeptide molecular weight of PHA-I determined by the light-scattering technique, considered together with the sequence molecular weights revealed for the subunits, indicated that PHA-I has the subunit stoichiometry of (alpha beta)2 complex. Inhibition test of PHA-I with increasing amounts of porcine pancreatic alpha-amylase (PPA) suggested that an inactive 2:1 complex is formed between PPA and PHA-I. In fact, two complexes differing from each other in the molar ratio of PPA to PHA-I were separated by gel filtration, and molecular weight estimation by the light-scattering technique confirmed that they are complexes of PHA-I with one or two PPA molecules. The binding of PPA to PHA-I appeared to follow simple binomial statistics, suggesting that two binding sites on PHA-I are independent and of high affinity for PPA.

Amino Acid Sequence↗
PubMed1995

Binding of Neu differentiation factor with the extracellular domain of Her2 and Her3.

The interaction of neu differentiation factor (NDF) with the extracellular domains of Her2 (sHer2) and Her3 (sHer3) have been studied using native gels, light scattering, and sedimentation equilibrium. The full-length NDF beta 2 was shown to bind sHer3 with a dissociation constant of 26 +/- 9 nM, while it showed a 1000-fold weaker binding to sHer2. Taken together, these results demonstrate that NDF is a high affinity ligand for Her3, but not for Her2. No increase in affinity of the NDF beta 2 for sHer3 was observed upon addition of sHer2 to the NDF beta 2-sHer3 mixture. Binding of NDF beta 2 to sHer3 did not induce receptor dimerization or oligomerization, the stoichiometry being one sHer3 per one NDF molecule. This finding suggests that transmembrane and/or intracellular domains of receptor family members or perhaps additional unidentified components may be involved in NDF induced dimerization and autophosphorylation, or alternatively, that dimerization is not the mechanism for Her3 autophosphorylation and signal transduction.

Animals↗
PubMed1995

Studies on the structure and function of glycosylated and nonglycosylated neu differentiation factors. Similarities and differences of the alpha and beta isoforms.

Comparative analyses of both glycosylated and nonglycosylated neu differentiation factor (NDF) isoforms revealed significant similarities and differences of their overall structures and functions. Biophysical analyses confirmed that all NDF isoforms are monomeric, but have an extended ellipsoidal shape in solution. All full-length NDFs are similar in secondary and tertiary structures and they contain no alpha-helix but are abundant in beta-strand structures. A small NDF fragment containing only the epidermal growth factor domain is also rich in beta-strand structures, but exhibits tertiary structure different from the long NDF forms. Monoclonal antibodies that selectively recognize epidermal growth factor domains of human NDF-alpha and -beta can specifically bind the respective NDF-alpha and -beta isoforms independent of NDF origins. Western blot analysis and quantitative binding assays further identify that an NDF preparation produced naturally from Rat1-EJ cells contains both alpha and beta isoforms in a 3 to 2 ratio. In receptor-binding competition experiments, human and rat NDF-beta isoforms have higher affinity than NDF-alpha isoforms. NDF-beta isoforms can dramatically enhance the stimulation of DNA synthesis for transfected NIH3T3 cells that overexpress HER-3 and HER-4 receptors, while NDF-alpha isoforms can only stimulate proliferation of HER-4-transfected cells with lower activity. Taken together, NDF-alpha and -beta isoforms share similar gross protein conformations but are biologically distinct.

3T3 Cells↗
PubMed1995

The importance of Arg40 and 45 in the mitogenic activity and structural stability of basic fibroblast growth factor: effects of acidic amino acid substitutions.

High-affinity binding of basic fibroblast growth factor (bFGF) to the tyrosine kinase receptor requires cell-surface heparan sulfate proteoglycan or exogenous addition of heparin. The crystal structure of bFGF shows Arg40 and 45 on the surface opposite to the heparin-binding region, suggesting that these charged residues may be involved in the receptor binding. Therefore, these amino acids were mutated to aspartic acid separately or simultaneously, and also a simultaneous mutation to glutamic acid was introduced. These mutants displayed a mitogenic activity decreased greater than tenfold compared to the wild-type protein. Addition of heparin had no effect on the activity, while these mutants showed heparin-binding characteristics resembling those of the native sequence protein. The mutants exhibited decreased stability compared to the native sequence protein. Gradual changes in conformation were observed by circular dichroic and infrared spectroscopy. Heparin chromatography also showed the presence of denatured form for these mutants. However, in the presence of multivalent anions such as citrate, sucrose octasulfate, and heparin, the conformation of the mutants resembled that of the wild-type protein, as revealed by X-ray crystallography and circular dichroism spectra of the mutant with a Arg40-->Asp substitution.

3T3 Cells↗
PubMed1994

Formation of heterodimers from three neurotrophins, nerve growth factor, neurotrophin-3, and brain-derived neurotrophic factor.

Three neurotrophic factors, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and nerve growth factor (NGF) form noncovalent homodimers in solution. Since they are highly homologous proteins, it seemed probable that two monomers of these proteins might associate together to form a heterodimer. This was tested by denaturing the two different proteins together in 6 M guanidine HCl and refolding them in phosphate-buffered saline. When the refolded mixture of BDNF and NT-3 was subjected to Mono S cation exchange chromatography, a new peak was observed eluting between NT-3 and BDNF, which accounted for about 30% of the protein used. This new protein species migrated as a single band upon native gel electrophoresis with mobility between that of the NT-3 homodimer and the BDNF homodimer, indicating that a complex had been formed. Sedimentation equilibrium data show that the dissociation constant of this heterodimer is < 3 x 10(-10) M. The heterodimer was stable upon incubation at 37 degrees C in phosphate-buffered saline over 11 days. Having determined that the heterodimer is highly stable, it was subjected to various biological assays. Autophosphorylation assay using TrkB receptor showed that the heterodimer is indistinguishable from the BDNF or NT-3 homodimer in the ability to induce phosphorylation of the receptor. It was also indistinguishable from the homodimers in the neurotrophic activity using chick dorsal root ganglion explant. In the sympathetic neuron survival assay, the heterodimer behaved more similarly to NT-3, whereas in the dopamine uptake assay, it was intermediate between the two homodimers. In addition, the heterodimer was shown to be retrogradely transported in the dorsal root ganglion neurons. A heterodimer between NGF and BDNF is formed but much less effectively than the NT-3.BDNF heterodimer, and it is not stable even at 4 degrees C. These results indicate that BDNF and NT-3 have an intersubunit contact surface for dimerization resembling each other's but different from the contact surface of NGF.

3T3 Cells↗
PubMed1994

Stoichiometry of heparin binding to basic fibroblast growth factor.

Fibroblast growth factors (FGFs) strongly bind to heparin and are thereby stabilized against deactivation and proteolytic cleavage. We have investigated the interactions of basic fibroblast growth factor (bFGF) with low- and high-molecular-weight heparin using size exclusion chromatography with on-line light scattering, absorbance, and refractive index detection. When heparin-bFGF mixtures with excess heparin are chromatographed using eluant that does not contain heparin, essentially all the protein is seen to elute as a complex with the heparin, indicating strong binding such that the complex does not dissociate significantly during chromatography (approximately 20 min). Combining the data from the light scattering, absorbance, and refractive index chromatograms allows us to determine the molecular weight of the protein component of the complex, and therefore to measure the number of bFGF molecules bound per heparin. A series of samples were prepared with a constant concentration of bFGF and variable amounts of a low-molecular-weight heparin (LMWH, M(r) = approximately 5000). At bFGF: heparin ratios above 1.5, a mix of complexes containing 3, 2, and 1 bFGF molecules is observed, with an average of 2.2 bFGF molecules per complex. Since the amount of bFGF incorporated into complexes implies an average of 2.5 +/- 0.3 bFGF molecules per heparin, there is only one heparin molecule per complex. The coexistence of complexes of different size when bFGF is in excess implies that the LMWH molecules are heterogeneous with respect to their ability to bind bFGF. When a high-molecular-weight heparin (HMWH, M(r) = 15,000) is used, complexes averaging 6.3 bFGF molecules per HMWH molecule are seen, while the overall amount of bFGF appearing in complexes implies six to seven sites per HMWH. These data show that the protein molecules can be packed very closely together. Both types of heparin give a heparin mass of 2300 Da per bFGF binding site, which corresponds approximately to an octasaccharide.

Chromatography, Gel↗
PubMed1993

Refolding of brain-derived neurotrophic factor from guanidine hydrochloride: kinetic trapping in a collapsed form which is incompetent for dimerization.

We have studied the pathway and kinetics of refolding of recombinant human brain-derived neurotrophic factor (BDNF), which is a very tightly-associated dimer in its native state. When BDNF unfolded in 6 M guanidine hydrochloride is diluted 20-fold into phosphate-buffered saline, a partially folded intermediate is rapidly formed (< 1 min). Circular dichroism and fluorescence spectroscopy show that this intermediate has extensive secondary structure, but no well-defined tertiary structure. Size-exclusion chromatography with light scattering detection shows that it is compact and monomeric, and therefore corresponds to what is often called a "collapsed form" or "molten globule". This collapsed form disappears with a half-time of approximately 30 min, simultaneously with the appearance of native dimers, without accumulation of monomeric species with a native tertiary structure. Remarkably, the monomer-dimer association constant of the collapsed form is approximately 10(10) weaker than the native structure, and it has a low tendency to form large aggregates. Given the very large hydrophobic surface present at the dimer interface of nerve growth factor (and presumably in BDNF), these results indicate that these hydrophobic groups are not exposed in the collapsed form, and that it is therefore quite dissimilar from the native structure. A significant conformational change in the collapsed form is necessary to re-expose these hydrophobic groups to form the dimer interface, making this the rate-limiting step in reaching the native conformation.

Brain-Derived Neurotrophic Factor↗
PubMed1993

Acid-induced unfolding of brain-derived neurotrophic factor results in the formation of a monomeric "a state".

Recombinant human brain-derived neurotrophic factor in acid undergoes a slow loss of tertiary structure as monitored by both near-UV circular dichroism and fluorescence, and appears to retain some secondary structure, as monitored by far-UV circular dichroism and Fourier transform infrared spectroscopy. This loss of tertiary structure parallels a decrease in the weight average molecular weight, from dimer to monomer, when examined using light scattering. Increasing the temperature accelerates this slow reaction. This process may be described most simply as N2 in equilibrium with 2D where N and D are the native and denatured forms of the protein, respectively. However, the acid denaturation strongly depends on the protein concentration, with higher concentration resulting in a lower rate and extent of denaturation. This suggests that the more complicated mechanism N2 in equilibrium with 2N in equilibrium with 2D more accurately describes the denaturation, where the dissociation into a native monomer is the rate-limiting step, and the conversion of N to D occurs relatively rapidly. Size-exclusion chromatography (at neutral pH) at several points during denaturation further demonstrated that the amount of tertiary structure remaining paralleled the dimer concentration and also that the monomer form was long-lived, remaining as monomer during the course of the chromatography. Size-exclusion chromatography and sedimentation velocity determination indicated that the acid-denatured form is a compact molecule. On the basis of the above data, the acid-denatured form may be considered to be a monomeric compact intermediate A state with no tertiary structure but considerable secondary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain-Derived Neurotrophic Factor↗
PubMed1993

Sites of iodination in recombinant human brain-derived neurotrophic factor and its effect on neurotrophic activity.

Recombinant human brain-derived neurotrophic factor (BDNF) is now under extensive investigation because of its potential clinical applications. Radioactively labeled proteins are usually required to study receptor binding and pharmacokinetic properties of proteins. This study was undertaken to see if iodination affects the biological and conformational properties of a recombinant BDNF. BDNF was iodinated using a stoichiometric amount of nonradioactive cold NaI to minimize multiple iodinations. Of the four tyrosines present in BDNF--Tyr-52, Tyr-54, Tyr-63, and Tyr-86--only Tyr-63 and Tyr-86 were iodinated under the experimental conditions used. Iodination of Tyr-63 resulted in modification without alteration of the biological activity, whereas iodination of Tyr-86 resulted in a molecule with highly compromised biological activity. Similar inactivation was observed if both Tyr-63 and Tyr-86 were iodinated. These modified proteins exhibited conformation and dimerization apparently identical to those of the native protein, as demonstrated by analytical ultracentrifugation, gel filtration, light scattering, and circular dichroism. From these results, we concluded that Tyr-52 and Tyr-54 are not accessible to the reagent and are probably buried in the hydrophobic core, whereas Tyr-63 and Tyr-86 are exposed on the surface of the molecule; of the two exposed residues, only Tyr-86 contributes to the biological activity.

Amino Acid Sequence↗